Ascorbate is the most abundant soluble antioxidant in plants and is also an essential nutrient for humans and a few other animals. Ascorbate contributes significantly to the overall intake of “free radical scavengers” or “anti-oxidative metabolites” in the human diet. Convincing evidence now shows that such metabolites either singly or in combination, benefit health and well-being, acting as anti-cancer forming agents and protecting against coronary heart disease.
Almost the entire dietary ascorbate intake in humans is derived from plant products. The ascorbate content of plant tissues however, is remarkably variable. Whilst leaf ascorbate content is generally high and relatively uniform in herbaceous and woody plants, a huge and unexplained variability in ascorbate content found is in non-green edible plant tissues. For example, in fruits, the levels vary from up to 30 mg gFW-1 AsA in the camu camu of Mirciaria dubia, to less than 3 μg gFW-1 AsA in the medlar of Mespilus germanica (Rodriguez et al. 1992, J Chromatogr Sci, 30:433-437). A range of values for ascorbate have been reported in kiwifruit (Ferguson, A. R., Botanical nominclature: Actinidia chinensis, Actinidia deliciosa, and Actinidia setosa. Kiwifruit: science and management, ed. I. J. Warrington and G. C. Weston. 1990, Palmerston North; New Zealand: New Zealand Society for Horticultural Science. 576. Beever, D. J. and G. Hopkirk, Fruit development and fruit physiology. Kiwifruit: science and management, ed. I. J. Warrington and G. C. Weston. 1990, Palmerston North; New Zealand: New Zealand Society for Horticultural Science. 576.) Ascorbate content of fruits from different vines range for A. deliciosa, 30-400 mg/100 g (Ferguson, A. R., 1991 Acta Hort. 290: p. 603-656, Spano, D., et al., 1997 Acta Hort., 444: p. 501-506.) while for the cultivar ‘Hayward’ the reported range is 80-120 mg/100 g (Beever, D. J. and G. Hopkirk, Fruit development and fruit physiology. Kiwifruit: science and management, ed. I. J. Warrington and G. C. Weston. 1990, Palmerston North; New Zealand: New Zealand Society for Horticultural Science. 576.). Higher concentrations of ascorbate are reported in fruit of, A. arguta, A. chinensis (Muggleston, S., et al., Orchardist, 1998. 71(8): p. 38-40, Chen, Q. and Q. Chen, Crop Genetic Resources, 1998(2): p. 3, Coggiatti, S., 1971 Ital Agr, October, 108(10): p. 935-941) A. chrysantha and A. polygama with very high levels in A. eriantha, and A. latifolia (>1% fresh weight) (Ferguson 1991 Acta Hort. 290: p. 603-656, and A. kolomikta (Kola, J. and J. Pavelka, 1988 Nahrung, 32(5): p. 513-515).
Three pathways of biosynthesis of ascorbic acid have been proposed in plants, one through L-Galactose (L-Gal) (Wheeler et al., 1998, Nature 393, 365-369), another from myo-Inositol (Loewus & Kelly, 1961, Arch. Biochem. Biophys. 95, 483-493; Lorence et al., (2004) Plant Physiol. 134, 1200-1205) and a third through Galacturonic acid (Agius et al., 2003, Nat Biotechnol 21, 177-81). The L-Gal pathway proceeds through L-Gal to galactono-1,4-lactone and thence to ascorbate (Wheeler et al., 1998, Nature 393, 365-369).
All the genes encoding enzymes, and their associated enzymatic activities, for the L-Galactose pathway have been identified and at least partially characterised.
The characterised genes and enzyme activities include the GDP-D-Mannose Pyrophosphorylase (Conklin, 1998, Trends Plant Sci 3: 329-330.; Conklin et al., 1999 Proc Natl Acad Sci USA 96: 4198-4203.; Keller et al., 1999 Plant J 19: 131-141.), the GDP-D-Mannose 3′,5′-Epimerase (Wolucka et al., 2001, Anal Biochem 294: 161-168; Wolucka and Van Montagu, 2003, J. Biol. Chem. 278: 47483-47490; Watanabe et al., 2006 Phytochemistry 67: 338-346.), the L-Galactose-1-P Phosphatase (Laing et al., 2004, Proceedings of the National Academy of Sciences (USA) 101: 16976-16981.; Conklin et al., 2006, J. Biol. Chem. 281: 15662-15670.), L-Galactose Dehydrogenase (Wheeler et al., 1998, Nature 393: 365-369.; Gatzek et al., 2002, Plant J. 30, 541 (2002; Laing et al., 2004 Proceedings of the National Academy of Sciences (USA) 101: 16976-16981), L-Galactono-1,4-lactone Dehydrogenase (Imai et al., 1998 Plant and Cell Physiology 39: 1350-1358.; Bartoli et al., 2005, Plant, Cell and Environment 28: 1073-1081.), and GDP-L galactose phosphorylase (GGP) (Laing et al., 2007, Proceedings of the National Academy of Sciences (USA) 104:9534-9). The applicants have previously shown that GDP-L galactose phosphorylase is central in determining ascorbate production Bulley S, et al 2012 Plant Biotechnol J 2012, 10:390-397.
Ascorbate concentrations are regulated according to demand. Under high light intensities when the need for high ascorbate is greatest, leaf ascorbate concentrations are raised (Bartoli et al., J. Exp. Bot. 57, 1621 (2006); Gatzek, et al., Plant J. 30, 541 (2002)). However little is known about the mechanism of regulation of ascorbate biosynthesis in plants (Bulley et al., Plant Biotechnol J 10, 390 (2012); Bulley et al., J. Exp. Bot. 60, 765 (2009).). Understanding how ascorbate biosynthesis is regulated may provide tools to manipulate biosynthesis in plants. Understanding the regulation of gene expression, and the factors/elements controlling such expression also provide valuable tools for genetic manipulation.
It is one object of the invention to provide improved compositions and methods for modulating GGP (also known as GDP-L-Galactose phosphorylase) activity; and/or ascorbate content in plants and/or to provide improved tools useful for genetic manipulation, or at least to provide the public with a useful choice.